PROJECT SUMMARY/ABSTRACT Tooth eruption is essential for survival of humans, with direct impact on the fundamental functions such as growth and development of the lower face, mastication for nutrition/energy intake, speech and esthetics for effective communication. Disorders involving tooth eruption are prevalent among the general public in a variety of forms such as delayed, ectopic and failure of eruption (2~4% for permanent molars). Many of these affected teeth do not respond well to orthodontic mechanical forces, often resulting in ankylosis. Extraction of these teeth results in morbidity and subsequently requires prostheses with inferior function, thus compromising patients' quality of life. Thus, more effective modalities to facilitate or reactivate tooth eruption are needed. The dental follicle (DF) surrounding the developing tooth coordinates tooth eruption and root formation. DF facilitates formation of osteoclasts that resorb alveolar bones to create the eruption pathway, and includes mesenchymal progenitor cells for cementoblasts and periodontal ligament (PDL) cells. These DF progenitor cells are crucial for development of periodontal tissues, which are particularly important for the later stage of tooth eruption. As loss-of-function mutations in PTH/PTHrP receptor (PPR) cause primary failure of tooth eruption (PFE) in humans, a PPR-dependent mechanism regulating DF progenitor cells is likely to play important roles for the coupling of tooth eruption and root formation. In this project, we hypothesize that DF progenitor cells are the central regulators of tooth eruption and root formation, whereby PTHrP-PPR signaling exerts important regulatory roles to maintain their unique properties of these cells. In Aim1, we will determine molecular characteristics of dental follicle progenitor cells in vivo. The working hypothesis is that DF cells express a unique set of transcription factors that maintain their properties as mesenchymal progenitor cells. Using the DF-specific PTHrP-creER transgenic line, we will define the gene expression profiles of individual DF progenitor cells based on a single cell RNA-seq analysis, followed by in situ validation of these genes. Further, we will establish a cell culture model to investigate heterogeneity and properties of DF progenitor cells through clonal selection and characterization. In Aim2, we will define roles of the PTH/PTHrP receptor in DF progenitor cells in tooth eruption. The working hypothesis is that PTHrP-PPR signaling in DF progenitor cells is essential to maintain their ability to self-renew and differentiate into periodontal ligament (PDL) cells. Using the mouse PFE model we have successfully established, we will determine the impact of PPR-deficiency on cell fate decision and self-renewal of DF progenitor cells using ex vivo cell culture systems. Further, using a comparative RNA-seq analysis, we will identify PPR-target genes responsible for the mouse PFE phenotypes in which tooth eruption is uncoupled from root formation. As a future research plan, we will further investigate key molecular mechanisms that regulate cell fate decisions of DF progenitor cells. We will particular focus on transcription factors downstream of PPR signaling.